Rotaviruses, members of the family Reoviridae, are the most important cause of severe acute diarrhea in infants and young children and on an annual basis are responsible for nearly one million deaths worldwide. Their medical significance has stimulated extensive and successful efforts towards the development of rotavirus vaccines by the Laboratory of Infectious Diseases (LID) at the NIH. Specifically, a rotavirus tetravalent vaccine developed by the LID has been shown in large scale phase III trials to have an efficacy of protection against severe and life-threatening dehydrating diarrhea of approximately 90%. The vaccine is less effective in preventing mild and moderate diarrhea stemming from rotavirus infection. The initial phase of this project focuses on the development of a reverse genetics system that will allow the introduction of genetic changes into the rotavirus genome. Subsequently this system will be used to identify regions of the genome that delineate the antigencity (serotype) and virulence of the virus. This information used in conjunction with the reverse genetics system will allow us to modify as necessary the antigenic properties of existing rotavirus vaccines so that they provide better protection against rotaviruses circulating in the population. Such a use for the reverse genetics system will be particularly important if circulating rotaviruses are found to undergo spontaneous mutation which causes a reduction in the effectiveness of the current LID vaccine in protecting against severe diarrhea. Furthermore, the reverse genetics system will also provide a mechanism to change the serotypes of the attenuated viruses that make up the vaccine. This may be important in regions of the world where the strains of rotavirus causing diarrheal disease are not of the same serotypes for which the current LID vaccine was designed to provide protection. In the final phase of the project, the reverse genetics system will be used to create new second generation vaccines through the introduction of attenuating mutations into the genome of virulent isolates of human rotaviruses. With the use of human rotaviruses as the basis for the development of new vaccines (as opposed to the current simian or bovine rotavirus based vaccines), it may be possible to generate rotavirus vaccines that protect not only against severe and life-threatening diarrhea but also provide better protection against mild and moderate diarrheal disease. Because of their likely wide-spread use throughout the world, the final phase of the project includes an analysis of the vaccines potential usefulness as a vector to protect children against other significant enteric pathogens including but not limited to toxigenic E. coli, hepatitis A virus, and the Norwalk family of human caliciviruses. Multiple experimental approaches will be tried concurrently to develope a rotavirus reverse genetics system. They are largely based on methods proven successful in other viral systems and all have the goal of introducing a cDNA-derived viral RNA containing genetic markers into the genome of infectious virus. These approaches include (i) transfection of cDNA-derived RNA transcripts into rotavirus-infected cells, (ii) transfection of T7 transcription vectors containing rotavirus cDNAs into rotavirus-infected cells expressing T7 RNA polymerase, (iii) introduction of cDNA-derived transcripts into infectious subviral particles recovered from infected cells, and (iv) assembly of infectious particles in vitro from either purified virion protein components or purified recombinant proteins.